Generally, the negative charge of a protein is increased when glutamine and asparagine residues in the protein are deamidated to generate carboxyl groups, resulting in a reduced isoelectric point and an increased hydrating ability. In addition, an increased static repelling force leads to a reduced interaction between proteins, i.e., a reduced associating property. Such changes allow the solubility and the water dispersibility of the protein to be increased greatly. Furthermore, the increased negative charge on the protein serves to unfold the protein conformation, changes the higher structure, and allows a hydrophobic region which was buried initially deep inside the molecule to be exposed on the surface of the molecule. Accordingly, the deamidated protein is imparted with an amphiphilic property, and serves as an ideal surfactant which gives a great improvement in the protein's emulsifying ability, emulsion stability, foaming performance and foam stability.
Thus, the deamidation of a protein leads to the improvement in various functions and characteristics of the protein, and the utility of the protein is far greatly enhanced (for example, see Molecular Approaches to Improving Food Quality and Safety, D. Chatnagar and T. E. Cleveland, eds., Van Nostrand Reinhold, New York, 1992, p.37).
Three methods for deamidating proteins enzymatically are known, including a protease treatment under a high pH (pH 10) condition (A. Kato, A. Tanaka, N. Matsudomi and K. Kobarashi, J. Agric. Food Chem., 35, 224, 1987), a transglutaminase method (M. Motoki, K. Seguro, A. Nio and K. Takinami, Agric. Biol. Chem., 50, 3025, 1986), and a peptide glutaminase method (UP 5082672A and J. S. Hamada and W. E. Marshall, J. Food Sci., 54, 598, 1989), which involve the following problems.
In the protease method, it is impossible to avoid a peptide bond cleavage, which is a primary reaction of the method, and by which the improvement in the function of the protein expected as a result of a deamidation is limited (especially the foam stability is reduced). It is also disadvantageous that a bitter taste occurs.
In the transglutaminase method, a crosslinking reaction as a result of an isopeptide bond formation between glutamine and lysine, which is a primary reaction of the method, should be suppressed by means of a preliminary chemical protection of an ε-amino group of the lysine residue. Accordingly, the production of an edible deamidated protein by this method requires a procedure in which a reversible protective group such as a citraconyl group is used previously as a protective group prior to the deamidation of glutamine and a subsequent deprotection should be followed further by the separation of a deamidated protein from liberated citraconic acid. Thus, this method is far less practical due to its complicated manufacturing process and poor cost efficiency as described above.
On the other hand, the peptide glutaminase method requires a protein hydrolysate (U.S. Pat. No. 5,082,672A and J. S. Hamada and W. E. Marshall, J. Food Sci., 54, 598, 1989) since the enzyme catalyzes mainly the deamidation of a peptide whose molecular weight is reduced and can not act as itself on a protein (M. Kikuchi, H. Hayashida, E. Nakano and K. Sakaguchi, Biochemistry, Vol. 10, 1222–1229, 1971 and B. P. Gill, A. J. O'Shaughnessey, P. Henderson and D. R. Headon, Ir. J. Food Sci. Technol., Vol. 9, 33–41, 1985). Thus, the concomitant use of a protease can not be avoided, and a bitter taste occurs as is experienced in the protease method described above, together with problematic peptide formation and reduction in functions, especially in the foam stability.
As a method for overcoming such problems, a method for deamidating a protein (JP-A-2000-50887) is disclosed which employs an enzyme which exerts its deamidation effect by acting directly on a protein (protein deamidating enzyme).
On the other hand, a method involving a protein denaturation by a treatment for example with heat or a denaturing agent is also known. The significance of the denaturation of a protein may for example be an improvement in the sensitivity of a protease, improvement in the digestibility, as well as an improvement in the protein functions such as the emulsification characteristics, foam characteristics and gelling characteristics. In the prior art, it is ordinary to denature a protein by a physical method or a chemical method. A physical method may for example be a heat treatment or a high pressure treatment. A chemical method may for example be a treatment with a denaturing agent (urea, guanidine hydrochloride), reducing agent, oxidant, acid and alkali.